Reactive oxygen species (ROS) can damage DNA, which has implications in the pathogenesis of numerous human diseases. Here in this proposal we hypothesize that intrastrand oxidative crosslink lesions at adjacent 5-methylcytosine guanine (mCG) site may contribute to CpG mutagenesis. In this respect, several crosslink lesions have been found at adjacent thymine guanine and cytosine guanine sites. Because cytosine, 5-methylcytosine and thymine have similar reactivity upon reaction with ROS as represented by hydroxyl radical attack and adjacent mCG and TG have similar geometry in B-form DNA, we predict that the types of crosslink lesions at TG sites should also form at mCG sites. The major thrust of this proposal is to demonstrate the formation of these crosslink lesions at mCG sites and to examine the structure-activity relationship of these crosslink lesions. To this end, we will employ synthetic organic chemistry to prepare photolabile reactive intermediates that are likely involved in the formation of the crosslink lesions. We will study the reactivity of these reactive intermediates in dinucleoside monophosphates, and single- and double-stranded oligodeoxynucleotides (ODNs) by analyzing the products evolving from the reaction. This will give us authentic crosslink lesion-bearing substrates in both dinucleoside monophosphates and ODNs. The former will be used for the identification and quantification of these lesions formed from normal DNA without the photolabile radical precursors. We will then use the lesion-bearing ODN substrates to examine the structural, thermodynamic, and mutagenic properties of these crosslink lesions. The outcome of the proposed research will provide important insights into the roles of these crosslink lesions in CpG mutagenesis.
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